H2603141计算溶液所需的质量、体积或浓度。
为什么选择此级别
，适用于对基线干扰要求严格的色谱和分析工作流程。
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文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
在分子生物学的试验过程中，
Fluo™ Green
® dsDNA 定量试剂盒是荧光检测双链 DNA 并进行定量 的一种产品，这种
方法非常灵敏。常用于分子生物学技术中的：cDNA 文库的构建；用于亚克隆的 DNA 片段纯化及应用，比如进行 DNA 定量、产物扩增和引物的进一步检测。 疫苗是现代疾病预防中常用的控制方式。如今许多疫苗是细胞培养疫苗，比如重组 乙肝疫苗、狂犬病疫苗等大多数疫苗都采用细胞培养的方法生产。其中，疫苗的纯化是关键问题，我们需要尽可能的去除 宿主细胞DNA和宿主蛋白。假若宿主细胞的DNA和蛋白同疫苗一起注入人体将会产生不可预料的后果。
常规的DNA含量的检测方法是在260nm（A260）处测其吸光值。这种方法的主要缺点是核苷酸、单链核酸和蛋白质对信 号的影响很大，并且还会受到核酸制备过程中污染物的干扰，无法区分DNA和RNA，而且这种方法不灵敏（5µg/mLdsDNA 溶液A260=0.1）。
Fluo™ Green
定量检测方法简单、方便，被多家生物制品厂所选择，成为生物制品残留DNA检测的标准。
目前该方法已纳入2010年版《中国药典》
原理:
Fluo™ Green
与DNA双链结合后才发出的荧光，无DNA不发荧光；所发荧光与DNA浓度成正比。在2010年《中国药典》中提 出，
Fluo™ Green
定量DNA的方法检出限约0.3ng/ml，DNA含量在1.25-80ng/mL范围时线性较好(R2>0.99).
优点：
1）该方法可以测定来源于任何表达宿主样品中的双链DNA。
2）可以直接定量PCR扩增产物而无需从反应混合物中纯化DNA。
3）远远超出传统紫外A260的检测方法和Hoechst33258的灵敏度。
4）较高浓度的盐，尿素，乙醇，氯仿，去垢剂，蛋白或琼脂糖对测定无影响。
5）在等摩尔浓度 ssDNA 和 RNA 存在的条件下测定 dsDNA，其影响很小。
所需器材
• 微型荧光计；便携式荧光仪-上海互帼科学仪器有限公司HG-9型；1cm石英比色皿
•
Fluo™ Green
dsDNA 定量检测试剂盒， 1mL 单位量的试剂浓缩液足够 2mL 体积的 200 次测定。
1×TE（10mM Tris 1mM EDTA）pH8.0； 250ug/mL 小牛胸腺DNA
实验方案
试剂制备
Fluo™ Green
dsDNA 定量试剂是以 1mL 的浓缩液形式保存在无水的 DMSO（二甲基亚砜）中。实 验当天，配制
2X
Fluo™ Green
试剂的操作溶液，用 1xTE 按 1:200 的比例稀释浓缩液（10mM Tris-HCl，
1mM EDTA，pH7.5）。如果要准备足够的操作溶液测定 20 个样品，可在 20mL1x TE 中加入 100µL
Fluo™ Green
dsDNA 定量试剂。由于试剂容易吸附到玻璃表面，要在塑料容器中配制。
Fluo™ Green
试剂 见光易降解，所以应将配 好的溶液用箔包住或放置暗处避光保存。
溶液最好在配制好数小时内使用，以保证最佳结果。
实验方法：
1). 标准品工作液的配制：
小牛胸腺嘧啶DNA干粉1mg（Tris，Nacl等浓度已成标准体系），加入1mL双蒸水， 配制成1mg⁄mL的标准品工作液；
2). 染料工作液的配置：
6 uL
Fluo™ Green
加入1mL TE(注意：用1×TE将
Fluo™ Green
稀释200倍，现用现配，注意避光)。
3). 标准品工作液稀释：
（1）母液稀释：取10ul（1mg⁄mL）标准品工作液加入到990ul TE溶液中，浓度稀释成10ug⁄mL，取10ul（10ug⁄mL） 标准品工作液加入到990ul TE溶液中，浓度稀释成100ng⁄mL；
（2）倍比稀释：取800ul （100ng⁄mL）的标准品工作液加入到200ul TE溶液中，浓度达到80ng⁄mL（药典规定：荧
光染色方法DNA含量在1.25-80 ng/mL范围线性较好， 该法DNA检出限为0.3 ng/ml），取500ul（80ng⁄mL）的标准品工 作液加入到 500ul TE溶液中，浓度稀释到 40ng⁄mL；依次倍比稀释， 配成 20ng/ml、 10ng/ml、 5.0ng/ml、 2.5ng/ml、
1.25ng/ml、0.625ng/ml的标准品溶液；
4).标准曲线的制备：倍比稀释后的各梯度标准品溶液和染料工作液各取100ul混匀，避光室温放置5min。使用FB-15型 便携式荧光仪检测样品的荧光值：将混合后的溶液加入微量比色皿，确信不要在样品中引入气泡，轻轻地弹微量检测皿 的外部，可以驱散气泡。以1×TE缓冲液为blank，测定样品和空白对照的荧光值；用标准品溶液的浓度（ng/ml）对应的
荧光强度作直线回归，制备标准曲线。
5). 测量剩余样品的荧光值。荧光计将给出一个直接的浓度读数，数据可以用来产生DNA 浓度的标准曲线。
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In the experimental process of molecular biology, Fluo ™ Green ® The dsDNA quantification kit is a product used for fluorescence detection and quantification of double stranded DNA
The method is very sensitive. Commonly used in molecular biology techniques: construction of cDNA libraries; Purification and application of DNA fragments for subcloning, such as DNA quantification, product amplification, and further detection of primers. Vaccines are a commonly used control method in modern disease prevention. Nowadays, many vaccines are cell culture vaccines, such as recombinant hepatitis B vaccine, rabies vaccine and most other vaccines are produced by cell culture. Among them, the purification of vaccines is a key issue, and we need to remove host cell DNA and host proteins as much as possible. If the DNA and protein of the host cell are injected into the human body together with the vaccine, unpredictable consequences will occur.
The conventional method for detecting DNA content is to measure its absorbance at 260nm (A260). The main disadvantage of this method is that nucleotides, single stranded nucleic acids, and proteins have a significant impact on the signal, and are also subject to interference from pollutants during the nucleic acid preparation process, making it difficult to distinguish between DNA and RNA. Additionally, this method is insensitive (5 µ g/mL dsDNA solution A260=0.1). Fluo ™ The Green quantitative detection method is simple and convenient, and has been selected by multiple biological product factories, becoming the standard for residual DNA detection in biological products.
At present, this method has been included in the 2010 edition of the Chinese Pharmacopoeia
Principle:
Fluo ™ Green emits fluorescence only after binding to double stranded DNA, and does not emit fluorescence without DNA; The fluorescence emitted is directly proportional to the concentration of DNA. In the 2010 Chinese Pharmacopoeia, it was proposed that, Fluo ™ The detection limit of Green's quantitative DNA method is about 0.3ng/ml, and the linearity is good (R2>0.99) when the DNA content is in the range of 1.25-80ng/mL
Advantage:
1) This method can determine double stranded DNA from any expressed host sample.
2) It is possible to directly quantify PCR amplification products without purifying DNA from the reaction mixture.
3) Far exceeding the sensitivity of traditional UV A260 detection methods and Hoechst33258.
4) Higher concentrations of salt, urea, ethanol, chloroform, detergents, proteins, or agarose have no effect on the measurement.
5) The effect of measuring dsDNA in the presence of equimolar concentrations of ssDNA and RNA is minimal.
Required equipment
• Micro fluorescence meter; Portable fluorescence analyzer - Shanghai Huguo Scientific Instrument Co., Ltd. HG-9 model; 1cm quartz colorimetric dish
• Fluo ™ Green dsDNA quantitative detection kit, 1mL of concentrated reagent solution is sufficient for 200 measurements of 2mL volume.
1×TE（10mM Tris 1mM EDTA）pH8.0； 250ug/mL calf thymus DNA
Experimental plan
Preparation of reagents
Fluo ™ The Green dsDNA quantification reagent is stored in anhydrous DMSO (dimethyl sulfoxide) in the form of 1mL concentrated solution. On the day of the experiment, prepare
2X Fluo ™ The operating solution of Green's reagent was diluted with 1xTE at a ratio of 1:200 in concentrated solution (10mM Tris HCl,
1mM EDTA, pH 7.5). If you want to prepare enough operating solution to determine 20 samples, you can add 100 µ L Fluo to 20mL1x TE ™ Green dsDNA quantification reagent. Due to the easy adsorption of reagents onto glass surfaces, they need to be prepared in plastic containers. Fluo ™ Green reagent is easily degraded by light, so the prepared solution should be wrapped in foil or stored in a dark place away from light.
It is best to use the solution within a few hours of preparation to ensure optimal results.
Experimental method:
1). Preparation of standard working fluid:
1mg of calf thymidine DNA dry powder (Tris, NaCl, and other concentrations have become standard systems) is added to 1mL of double distilled water to prepare a 1mg/mL standard working solution;
2). Configuration of dye working fluid:
6 uL Fluo ™ Add 1mL of TE to Green (note: use 1 × TE to mix Fluo) ™ Dilute Green 200 times, use and prepare immediately, avoid light.
3). Dilution of standard working fluid:
(1) Mother liquor dilution: Take 10ul (1mg ⁄ mL) of standard working solution and add it to 990ul TE solution to dilute the concentration to 10ug ⁄ mL. Take 10ul (10ug ⁄ mL) of standard working solution and add it to 990ul TE solution to dilute the concentration to 100ng ⁄ mL;
(2) Dilute by multiple ratio: Take 800ul (100ng ⁄ mL) of standard working solution and add it to 200ul of TE solution to achieve a concentration of 80ng ⁄ mL (pharmacopoeia regulation: fluorescent)
The light staining method shows good linearity in the range of 1.25-80 ng/mL for DNA content, with a detection limit of 0.3 ng/mL. Take 500ul (80ng ⁄ mL) of standard working solution and add it to 500ul TE solution, diluting the concentration to 40ng ⁄ mL; Dilute sequentially by multiple ratios to prepare 20ng/ml 10ng/ml 5.0ng/ml 2.5ng/ml
1.25ng/ml and 0.625ng/ml standard solution;
4). Preparation of standard curve: Take 100ul of each gradient standard solution and dye working solution diluted by multiple ratios, mix well, and leave them at room temperature in the dark for 5 minutes. Use FB-15 portable fluorescence analyzer to detect the fluorescence value of the sample: Add the mixed solution to the microcalorimeter, make sure not to introduce bubbles into the sample, and gently tap the outside of the microcalorimeter to disperse the bubbles. Measure the fluorescence values of the sample and blank control using 1 × TE buffer as a blank; Corresponding to the concentration of standard solution (ng/ml)
Perform linear regression on fluorescence intensity and prepare a standard curve.
5). Measure the fluorescence value of the remaining samples. The fluorescence meter will provide a direct concentration reading, which can be used to generate a standard curve of DNA concentration.
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规格
英文名称
Fluo™ Green dsDNA Assay Kit
储存与运输
储存条件
避光,-20°C储存,干燥
运输条件
超低温运输
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
溶液计算器
常见问题
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